Three different genes encode the iron-sulfur subunit of succinate dehydrogenase in Arabidopsis thaliana.

The iron-sulfur protein is an essential component of mitochondrial complex II (succinate dehydrogenase, SDH), which is a functional enzyme of both the citric acid cycle and the respiratory electron transport chain. This protein is encoded by a single-copy nuclear gene in mammals and fungi and by a mitochondrial gene in Rhodophyta and the protist Reclinomonas americana. In Arabidopsis thaliana, the homologous protein is now found to be encoded by three nuclear genes. Two genes (sdh2-1 and sdh2-2) likely arose from a relatively recent duplication event since they have similar structures, encode nearly identical proteins and show similar expression patterns. Both genes are interrupted by a single intron located at a conserved position. Expression was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the third gene (sdh2-3) is interrupted by 4 introns, is expressed at a low level, and encodes a SDH2-3 protein which is only 67% similar to SDH2-1 and SDH2-2 and has a different N-terminal presequence. Interestingly, the proteins encoded by these three genes are probably functional because they are highly conserved compared with their homologues in other organisms. These proteins contain the cysteine motifs involved in binding the three iron-sulfur clusters essential for electron transport. Furthermore, the three polypeptides are found to be imported into isolated plant mitochondria.

A nuclear casein kinase 2 activity is involved in early events of transcriptional activation induced by salicylic acid in tobacco.

Salicylic acid (SA) activates immediate early transcription of genes controlled by a family of DNA promoter elements named as-1-like elements. These elements are functional in the promoter of glutathione S-transferase genes. We have previously shown that SA increases the binding of tobacco (Nicotiana tabacum cv Xanthi nc) nuclear factors to the as-1 sequence in a process mediated by protein phosphorylation. In this study we give evidence for the participation of a nuclear protein kinase CK2 (casein kinase 2) in the pathway activated by SA in tobacco. The first line of evidence comes from the evaluation of the CK2 activity in nuclear extracts prepared from tobacco plants treated with SA or water as a control. Results from these experiments indicate that SA increases the nuclear CK2 activity. The second line of evidence derives from the evaluation of the in vivo effect of 5,6-dichloro-1-(beta-D-ribofuranosyl) benzimidazole (DRB), a cell-permeable CK2 inhibitor, on the responsiveness of the as-1 sequence to SA. Results from these experiments indicate that DRB impairs the activating effect of SA on the transcription of both, the GUS reporter gene controlled by a tetramer of the as-1 element, and the endogenous gnt35 gene encoding a glutathione S-transferase, in transgenic tobacco plants. DRB also impaired the increasing effect of SA on the binding of nuclear factors to the as-1 element. Furthermore, transcription of the as-1/GUS reporter gene activated by the synthetic auxin 2,4-dichlorophenoxyacetic acid and by methyl jasmonate was also inhibited by DRB. To our knowledge, this is the first report in which activation of a CK2 enzyme by a plant hormone is reported.

Cloning and characterization of the cDNA coding for the catalytic alpha subunit of CK2 from tobacco.

We have previously reported the participation of the protein kinase CK2 in the mechanism by which salicylic acid activates transcription of genes, such as those coding for glutathion S-transferases, in tobacco. With the purpose of further studying the participation of CK2 in this signal transduction pathway, we isolated and sequenced the cDNA from the NtCK2A gene, coding for the catalytic alpha subunit of CK2 from tobacco. The NtCK2A cDNA was isolated by screening of a tobacco cDNA library with a heterologous probe from Arabidopsis thaliana, followed by 3' RACE to obtain the 3' region. Sequence analysis of the NtCK2A cDNA showed a high level of identity between this CK2alpha protein sequence and the corresponding sequences of other plant species such as Arabidopsis and maize (92-95% identity), or those of animal species such as human and Xenopus laevis (75% identity). The expression of the NtCK2A gene in different tissues from tobacco plants was analyzed by Northern blot. High levels of expression of this gene were observed in proliferating tissues such as shoot and root apical meristems. A recombinant CK2alpha protein was obtained after expression of the NtCK2A cDNA in Escherichia coli. The ability of this recombinant CK2alpha subunit to phosphorylate casein was inhibited by heparin and stimulated by the CK2beta subunit from Xenopus laevis.

The nuclear-encoded SDH2-RPS14 precursor is proteolytically processed between SDH2 and RPS14 to generate maize mitochondrial RPS14.

In maize, the functional gene encoding mitochondrial ribosomal protein S14 (rps14) has been translocated to the nucleus where it became integrated between both exons of a gene encoding the iron-sulfur subunit of succinate dehydrogenase (sdh2). Two transcripts are generated from this locus by alternative splicing. One transcript encodes a precursor for a functional SDH2 protein, while the second transcript encodes a chimeric SDH2(t)-RPS14 precursor protein. In this paper we show that the same mitochondrial targeting presequence is able to direct the import of both precursors into isolated mitochondria and is removed during import. This processing event generates a 28 kDa protein from the SDH2 precursor, which corresponds to the iron-sulfur subunit of respiratory complex II present in maize mitochondria. In addition to cleavage of the presequence, the chimeric precursor undergoes proteolytical processing between SDH2 and RPS14. This processing generates RPS14, which is found assembled into mitochondrial ribosomes, and a truncated SDH2 protein which is degraded. Therefore, our results support a role of the SDH2 domain in the chimeric precursor only in providing a mitochondrial targeting function for RPS14.

Signal transduction networks and the biology of plant cells.

The development of plant transformation in the mid-1980s and of many new tools for cell biology, molecular genetics, and biochemistry has resulted in enormous progress in plant biology in the past decade. With the completion of the genome sequence of Arabidopsis thaliana just around the corner, we can expect even faster progress in the next decade. The interface between cell biology and signal transduction is emerging as a new and important field of research. In the past we thought of cell biology strictly in terms of organelles and their biogenesis and function, and researchers focused on questions such as, how do proteins enter chloroplasts? or, what is the structure of the macromolecules of the cell wall and how are these molecules secreted? Signal transduction dealt primarily with the perception of light (photomorphogenesis) or hormones and with the effect such signals have on enhancing the activity of specific genes. Now we see that the fields of cell biology and signal transduction are merging because signals pass between organelles and a single signal transduction pathway usually involves multiple organelles or cellular structures. Here are some examples to illustrate this new paradigm. How does abscisic acid (ABA) regulate stomatal closure? This pathway involves not only ABA receptors whose location is not yet known, but cation and anion channels in the plasma membrane, changes in the cytoskeleton, movement of water through water channels in the tonoplast and the plasma membrane, proteins with a farnesyl tail that can be located either in the cytosol or attached to a membrane, and probably unidentified ion channels in the tonoplast. In addition there are highly localized calcium oscillations in the cytoplasm resulting from the release of calcium stored in various compartments. The activities of all these cellular structures need to be coordinated during ABA-induced stomatal closure. For another example of the interplay between the proteins of signal transduction pathways and cytoplasmic structures, consider how plants mount defense responses against pathogens. Elicitors produced by pathogens bind to receptors on the plant plasma membrane or in the cytosol and eventually activate a large number of genes. This results in the coordination of activities at the plasma membrane (production of reactive oxygen species), in the cytoskeleton, localized calcium oscillations, and the modulation of protein kinases and protein phosphatases whose locations remain to be determined. The movement of transcription factors into the nucleus to activate the defense genes requires their release from cytosolic anchors and passage through the nuclear pore complexes of the nuclear envelope. This review does not cover all the recent progress in plant signal transduction and cell biology; it is confined to the topics that were discussed at a recent (November 1998) workshop held in Santiago at which lecturers from Chile, the USA and the UK presented recent results from their laboratories.

The gene for mitochondrial ribosomal protein S14 has been transferred to the nucleus in Arabidopsis thaliana.

The transfer of genetic information from the mitochondrion to the nucleus is thought to be still underway in higher plants. The mitochondrial genome of Arabidopsis thaliana contains only one rps14 pseudogene. In this paper we show that the functional gene encoding mitochondrial ribosomal protein S14 has been translocated to the nucleus. This gene transfer is a recent evolutionary event, which occurred within Cruciferae, probably after the divergence of Arabidopsis and Brassica napus. A 5' extension of the rps14 reading frame encodes a presequence which, in vitro, targets the polypeptide to isolated mitochondria and is cleaved off during or after import. No intron was found at the junction of the targeting presequence with the mitochondrially derived sequence, which are directly connected. By contrast, a 90-bp intron, which is removed by splicing to give a mature poly(A)+ mRNA of 0.9 kb, is located in the 3' non-coding region. To our knowledge, this is the first report of an intron in such a position in a functional transferred gene in higher plants, and suggests that exon shuffling may have been involved in the acquisition of elements necessary for expression in the nucleus. Putative roles of this intron in polyadenylation and enhancement of gene expression are discussed.

Transfer of rps14 from the mitochondrion to the nucleus in maize implied integration within a gene encoding the iron-sulphur subunit of succinate dehydrogenase and expression by alternative splicing.

The maize mitochondrial genome does not contain a gene coding for ribosomal protein S14. In this paper we show that the functional rps14 gene was translocated to the nucleus and acquired the signals conferring expression and product targeting to the mitochondrion in a way not previously described. Transferred rps14 was found integrated between both exons of a gene encoding the iron-sulphur subunit of the respiratory complex II (sdh2). Sdh2 exon 1 and rps14 were separated by a typical plant nuclear intron that was spliced to give a mature poly(A)+ mRNA of 1.4 kb. This processed mRNA encoded a chimeric SDH2 (truncated)-RPS14 polypeptide, and we show that this chimeric polypeptide is targeted into isolated plant mitochondria, where it is proteolytically processed in a complex way. An alternative splicing event utilizing the same 5' splice site and a different downstream 3' splice site generated a second mature poly(A)+ mRNA of 1.3 kb that contained both sdh2 exons. This sdh2 transcript encoded an SDH2 polypeptide highly conserved compared with its homologues in other organisms, and it contained the three cysteine-rich clusters that made up the three non-heme iron-sulphur centres responsible for electron transport. To our knowledge, these results constitute the first evidence of alternative splicing playing a role in the expression and targeting of two mitochondrial proteins with different functions from the same gene.

Editing status of mat-r transcripts in mitochondria from two plant species: C-to-U changes occur in putative functional RT and maturase domains.

The intronic mat-r ORF encodes a protein with significant homology to retroviral reverse transcriptases. Here, we describe the nucleotide sequence of potato mat-r and study the editing status of mat-r transcripts in two systems, potato and wheat, where the mat-r ORF is part of the trans-introns but in two different configurations relative to nad1 exons d and e. In potato and wheat, 13 and 15 C-to-U transitions respectively were observed. Most transcripts were partially edited, but potato transcripts were edited more efficiently than wheat transcripts. As in functional mitochondrial genes, RNA editing increased the similarity between plant mat-r proteins and their homologous non-plant counterparts. Interestingly, editing of mat-r was clustered in the reverse-transcriptase (RT) and the maturase (X) domains, two well defined regions having known functions in other systems. These results, together with the integrity and sequence conservation of mat-r, strongly suggest that the encoded protein plays a functional role in plant mitochondria.

Phosphorylation of nuclear proteins directs binding to salicylic acid-responsive elements.

The cis-located DNA sequence as-1 (Activation Sequence-1) from CaMV 35S promoter has been previously identified as an element that can confer inducibility by salicylic acid (SA) with immediate early kinetics. This sequence specifically binds to ASF-1 (Activation Sequence Factor-1), previously characterized in tobacco nuclear extracts. To assess whether modulation of ASF-1 binding activity can explain the activation of the as-1 sequence observed in vivo, we performed electrophoretic mobility shift assays using nuclear extracts from SA-treated and water-treated tobacco plants. Our results indicate that treatment of plants with SA increases ASF-1 binding to as-1 and to ocs, an as-1-like element from the Agrobacterium octopine synthase gene. In contrast, SA treatment has no effect on the binding of GT-1 factor to its target light-inducible box II element. Furthermore, treatment of nuclear extracts from SA-treated plants with alkaline phosphatase decreases ASF-1 binding to the as-1 element. This can be reversed by pretreatment with 10 mM NaF. Accordingly, pretreatment of nuclear extracts from control water-treated plants with ATP produces an increase in ASF-1 binding activity similar to that observed with SA. This effect of ATP is reversed by treatment with alkaline phosphatase and prevented by quercetin, a casein kinase II inhibitor. These results support the hypothesis that a nuclear protein kinase is involved in the immediate early events of transcriptional activation triggered by SA.

The rpl5-rps14-cob gene arrangement in Solanum tuberosum: rps14 is a transcribed and unedited pseudogene.

The L5 ribosomal protein gene (rpl5) and a S14 ribosomal protein pseudogene were identified by sequence analysis in the potato mitochondrial genome. The two genes are separated by one nucleotide and are found upstream of the apocytochrome b gene (cob), an arrangement conserved also in Arabidopsis and Brassica. The rpl5 gene has an intact open reading frame while the rps14 locus is disrupted by a five nucleotide duplication that introduces a frameshift in the reading frame. Editing of rpl5 and pseudorps14 cotranscripts has been studied by cDNA sequence analysis. Eight C residues are edited into U in the rpl5 coding region, resulting in eight amino acid changes that increase the homology between potato and other RPL5 polypeptides. Interestingly, the rps14 pseudogene sequence is not edited at any nucleotide position.

Splicing and editing of rps10 transcripts in potato mitochondria.

The structure and expression of the potato mitochondrial gene rps10, encoding ribosomal protein S10, has been characterized. The RPS10 polypeptide of 129 amino acids is encoded by two exons of 307 bp and 80 bp respectively, which are separated by a 774-bp class-II intron. Editing of the complete rps10 coding region was studied by sequence analysis of spliced cDNAs. Four C residues are edited into U, resulting in the creation of a putative translational initiation codon, a new stop codon which eliminated ten carboxy-terminal residues, and two additional amino-acid alterations. All these changes increase the similarity between the potato and liverwort polypeptides. One additional C-to-U RNA editing event, observed in the intron sequence of unspliced cDNAs, improves the stability of the secondary structure in stem I (i) of domain I and may thus be required for the splicing reaction. All spliced cDNAs, and most unspliced cDNAs, were completely edited, suggesting that editing is an early step of rps10 mRNA processing and precedes splicing. Earlier work on potato rps10 (Zanlungo et al. 1994) is now known to comprise only a partial analysis of the gene, since the short downstream exon was not identified.

Transcription initiation sites for the potato mitochondrial gene coding for subunit 9 of ATP synthase (atp9).

The potato mitochondrial atp9 gene has a simple expression pattern. To determine where transcription initiates, primary mitochondrial RNAs were labeled by in vitro capping and hybridized to the 5' flanking sequences of the atp9 gene. A single transcription initiation region was identified. Primer extension and nuclease S1 protection analyses were used to precisely map the transcript 5' termini in this region. These results indicate that transcription initiates at 121-128 bp upstream of the atp9 open reading frame, in a sequence which does not present any homology with proposed consensus sequences for plant mitochondrial promoters. Nuclease S1 protection were also used to map 3' termini 67-71 nucleotides downstream of a putative single-stem loop structure.

A ribosomal protein S10 gene is found in the mitochondrial genome in Solanum tuberosum.

The S10 ribosomal protein gene (rps10), which has not been previously reported in any angiosperm mitochondrial genome, was identified by sequence analysis in the potato mitochondrial DNA. This gene is found downstream of a truncated non-functional apocytochrome b (cob) pseudogene, and is expressed as multiple transcripts ranging in size from 0.8 to 5.0 kb. Southern hybridization analysis indicates that rps10-homologous sequences are not present in the wheat mitochondrial genome. Sequence analysis of a single-copy region of the pea mitochondrial genome located upstream of cox1 [11] shows that a non-functional rps10 pseudogene is present in this species. These results suggest that the functional genes coding for wheat and pea mitochondrial RPS10 polypeptides have been translocated to the nucleus.

Immediate early transcription activation by salicylic acid via the cauliflower mosaic virus as-1 element.

Transgenic tobacco plants carrying a number of regulatory sequences derived from the cauliflower mosaic virus 35S promoter were tested for their response to treatment with salicylic acid (SA), an endogenous signal involved in plant defense responses. beta-Glucuronidase (GUS) gene fusions with the full-length (-343 to +8) 35S promoter or the -90 truncation were found to be induced by SA. Time course experiments revealed that, in the continuous presence of SA, the -90 promoter construct (-90 35S-GUS) displayed rapid and transient induction kinetics, with maximum RNA levels at 1 to 4 hr, which declined to low levels by 24 hr. Induction was still apparent in the presence of the protein synthesis inhibitor cycloheximide (CHX). Moreover, mRNA levels continued to accumulate over 24 hr rather than to decline. By contrast, mRNA from the endogenous pathogenesis-related protein-1a (PR-1a) gene began to accumulate at later times during SA treatment and steadily increased through 24 hr; transcription of this gene was almost completely blocked by the presence of CHX. Further dissection of the region from -90 and -46 of the 35S promoter revealed that the SA-responsive element corresponds to the previously characterized activation sequence-1 (as-1). These results represent a definitive analysis of immediate early responses to SA, relative to the late induction of PR genes, and potentially elucidate the early events of SA signal transduction during the plant defense response.

Ciprofibrate, a carcinogenic peroxisome proliferator, increases the phosphorylation of epidermal-growth-factor receptor in isolated rat hepatocytes.

Ciprofibrate, a hypolipidaemic drug with carcinogenic and peroxisome-proliferation effects in rat liver, was found to increase the phosphorylation of epidermal-growth-factor receptor in 32P-labeled isolated rat hepatocytes. This effect was suppressed by protein-kinase-C inhibitors, and was accompanied by an almost complete inhibition of the receptor autophosphorylation normally induced by its ligand. However, in vitro experiments showed that protein-kinase-C phosphorylation of purified epidermal-growth-factor receptor was activated by ciprofibroyl-CoA, the acyl-CoA derivative of the drug, but not by the unmodified drug. Neither compound affected the ligand induction of epidermal-growth-factor-receptor autophosphorylation in isolated liver membranes. These results suggest that metabolically produced ciprofibroyl-CoA in liver cells would activate protein-kinase-C and produce changes in epidermal-growth-factor-receptor function.

Epidermal growth factor receptor in synaptic fractions of the rat central nervous system.

Functional relationships between epidermal growth factor (EGF) and neural tissues have of late attracted increasing interest. However, in spite of reported EGF effects on neurons, the expression of the EGF receptor (EGF-R) has not yet been unambiguously demonstrated in these cells. This 170-kDa protein bears an intracellular tyrosine kinase domain in which activity is ligand-dependent. We give definitive evidence here for its presence in neonatal and adult rat neurons showing also, for the first time, its binding and functional tyrosine kinase activities in the synaptic region. Immunohistochemistry using a polyclonal antibody prepared against the receptor purified from rat liver showed positive staining localized exclusively to neurons without regionalization to any particular brain zone. Binding studies made in Percoll-obtained synaptosomes revealed specific high affinity 125I-EGF binding sites (Kd, 1.42 x 10(-10) +/- 0.58 M) accounting for 17% of total binding and a great majority of low affinity (Kd, 2.55 x 10(-9) +/- 0.35 M) binding sites. Higher binding capacity was found in synaptosomal fractions obtained from newborn rats. The identity of the synaptosomal EGF binding activity with the 170-kDA EGF-R protein was demonstrated by cross-linking experiments. Furthermore, EGF-Affi-Prep affinity chromatography adsorbs a 170-kDa protein with EGF-R immunoreactivity from whole homogenates of adult rat brain. Phosphorylation assays made in freeze-thawed or intact synaptosomes showed EGF-induced tyrosine phosphorylation in the range of 170-, 126-150-, 124-, 113-, 98-, and 70-kDa proteins including the EGF-R. Thus, the EGF-R/EGF regulatory system could have a role in synaptic function that remains to be explored.

Evidence of tyrosine kinase activity in the photosynthetic bacterium Rhodospirillum rubrum.

The photosynthetic bacterium Rohodospirillum rubrum evidenced tyrosine protein phosphorylation under photoautotrophic conditions in the presence of [32P]Pi. The stability to alkaline treatment of the [32P] bound to the cell-free extract proteins suggested that tyrosine residues were carrying the labelling. One- and two-dimensional high voltage paper electrophoresis analysis revealed that such extracts do contain [32P]-phosphotyrosine residues. Furthermore, the association of alkali stable [32P] bound to specific proteins of the cell-free extract was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with KOH treatment of the gel. A definite argument in favor of protein kinase(s) phosphorylating tyrosine residues in R.rubrum proteins was obtained by partial purification of a tyrosine kinase activity from cell-free extract capable of phosphorylating synthetic peptides that only contain a single tyrosine residue as phosphate acceptor.